Mutation of a single amino acid (T45I) leads to insensitivity to Vpu and persistence of tetherin protein.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the absence of HIV-1 Vpu (black bar), or presence (white bar). Mutation T45I in the trans-membrane region of human tetherin results in insensitivity to Vpu whilst maintaining antiviral activity. The titre of the unrestricted HIV-1 was 10⁷ infectious units/ml. Errors are standard error of the mean of 2 experiments. (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in cleared RIPA extracts from transfected 293T cells. Tetherin was detected by western blot of N-terminal Xpress tag in the cleared RIPA extract supernatants (D) and pellets (F) as shown. Sizes of molecular weight markers are shown in kilodaltons. Blots in (E) and (G) have been stripped and re-probed for β actin as a loading control. Data are representative of 2 independent experiments.</p

Diversity measured as average pairwise distance at longitudinal time points.

<p>APD measured pre- ASCT and during subsequent viral rebounds. Numbers in parentheses indicate the number of single genomes acquired at each time point. Significance was determined by two-tailed.</p

Selection analyses reveal positively selected tetherin residues in the primate lineage.

<p>(A) Nucleotide alignment of nine primate tetherin sequences. HoSa (Homo Sapiens), Popy (Pongo Pygmaeus (Orangutan)) Patr (Pan Trolodytes (Chimpanzee)), Tan (Tantalus monkey), Ver (Vervet monkey), Mane (Macaque Nemestrina (pigtailed macaque)), Mafa (Macaque Fasicularis (cynomolgus monkey)), Mamu (Macaque Mulatta (Rhesus macaque)) Caja (Callithrix jacchus (white tufted ear marmoset). Codon positions under positive selection are indicated by shaded boxes. Secondary structure (SS) was predicted by PSIPRED <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000443#ppat.1000443-McGuffin1" target="_blank">[53]</a> and is symbolised as (+) cytoplasmic domain; (I) trans-membrane domain inner cap; (X) trans-membrane domain alpha helix; (O) trans-membrane domain outer cap; (−) extra-cellular domain. (B) Predicted structure of the trans-membrane helix performed using helical wheel projection (<a href="http://rzlab.ucr.edu/scripts/wheel/wheel.cgi" target="_blank">http://rzlab.ucr.edu/scripts/wheel/wheel.cgi</a>) suggests that residues 26, 30, and 36 are on the same side of the protein. Closed circles indicate positively selected amino acids, dashed circles indicate residues that are different between human and Tantalus monkey, but not positively selected. Human amino acids are shown in black and Tantalus monkey in grey italic.</p

Non-parametric geographic subdivision tests comparing pre- ASCT viruses with those at each rebound.

<p>Non-parametric geographic subdivision tests comparing pre- ASCT viruses with those at each rebound.</p

HIV-1 Vpu–mediated loss of tetherin is partially abrogated by MG132.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the presence or absence of HIV-1 Vpu-HA co-expression and proteasome inhibitor MG132 (0.8 µM for 12 hours) as shown. Errors are standard error of the mean of 2 experiments. (B) Tetherin was detected by western blot of Xpress tag in cleared RIPA extract supernatants and pellets as shown. Sizes of molecular weight markers are shown in kilodaltons. (C) Vpu-HA was detected in sonicated RIPA extract as shown (D) Blots in B have been stripped and re-probed for β actin as a loading control. (E) Measurement of HIV-1 p24 in the supernatant of transfected cells by western blot. Data are representative of 2 independent experiments.</p

Inferred neighbour-joining phylogenetic tree of single genome derived HIV-1 env sequences.

<p>Two APOBEC hypermutated sequences found in the third rebound have been removed. Pre melphalan HIV DNA (-27 to -38, orange open diamonds), first rebound HIV RNA (+6, blue closed diamonds), first rebound HIV DNA (+6, blue open diamonds), second rebound HIV RNA (+515, magenta closed diamonds), third rebound HIV DNA (+643 to +958, green open diamonds). Rooted on MJ4 (black square), branches with bootstrap support > 75% are indicated by black asterisk. Overall population APD is 1.5%. The scale represents 0.005 nucleotide substitutions per site, equivalent to 4.5nt. The tips in the boxed grey area representative sequences predicted to confer CXCR4 tropism by both Geno2Pheno and Phenoseq genotypic algorithms.</p

The positively selected tetherin trans-membrane region residues impact on sensitivity to HIV-1 Vpu.

<p>(A) Co-expression of HIV-1 plasmids alone (C) with wild-type human tetherin (WT) and measurement of HIV-1 release in the absence of HIV-1 Vpu (white bar) or presence (black bar). Mutation of positively selected residues I26V, V30G, I36L, T45I (Quad) in the trans-membrane region of human tetherin results in reduced sensitivity to Vpu whilst maintaining similar antiviral activity. The effect of single mutations are also shown. Errors are standard error of the mean of 2 experiments. Equal amounts of tetherin plasmids were used (100 ng) (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in extracts from transfected 293T cells. (D) Cell extract blots in C were stripped and re-probed for β actin as a loading control.</p

Genotypically defined co-receptor usage following HIV rebounds.

<p>Tropism testing of the HIV V3 loop was performed in both Geno2Pheno and Phenoseq online genotypic algorithms. Percentage of CXCR4 sequences is plotted on the Y-axis. Only sequences found to be X4 in both programs were designated as X4. Significance was determined using a two-tailed Fishers Exact test.</p

Treatment eligibility and retention in clinical HIV care: A regression discontinuity study in South Africa

<div><p>Background</p><p>Loss to follow-up is high among HIV patients not yet receiving antiretroviral therapy (ART). Clinical trials have demonstrated the clinical efficacy of early ART; however, these trials may miss an important real-world consequence of providing ART at diagnosis: its impact on retention in care.</p><p>Methods and findings</p><p>We examined the effect of immediate (versus deferred) ART on retention in care using a regression discontinuity design. The analysis included all patients (<i>N</i> = 11,306) entering clinical HIV care with a first CD4 count between 12 August 2011 and 31 December 2012 in a public-sector HIV care and treatment program in rural South Africa. Patients were assigned to immediate versus deferred ART eligibility, as determined by a CD4 count < 350 cells/μl, per South African national guidelines. Patients referred to pre-ART care were instructed to return every 6 months for CD4 monitoring. Patients initiated on ART were instructed to return at 6 and 12 months post-initiation and annually thereafter for CD4 and viral load monitoring. We assessed retention in HIV care at 12 months, as measured by the presence of a clinic visit, lab test, or ART initiation 6 to 18 months after initial CD4 test. Differences in retention between patients presenting with CD4 counts just above versus just below the 350-cells/μl threshold were estimated using local linear regression models with a data-driven bandwidth and with the algorithm for selecting the bandwidth chosen ex ante. Among patients with CD4 counts close to the 350-cells/μl threshold, having an ART-eligible CD4 count (<350 cells/μl) was associated with higher 12-month retention than not having an ART-eligible CD4 count (50% versus 32%), an intention-to-treat risk difference of 18 percentage points (95% CI 11 to 23; <i>p <</i> 0.001). The decision to start ART was determined by CD4 count for one in four patients (25%) presenting close to the eligibility threshold (95% CI 20% to 31%; <i>p <</i> 0.001). In this subpopulation, having an ART-eligible CD4 count was associated with higher 12-month retention than not having an ART-eligible CD4 count (91% versus 21%), a complier causal risk difference of 70 percentage points (95% CI 42 to 98; <i>p <</i> 0.001). The major limitations of the study are the potential for limited generalizability, the potential for outcome misclassification, and the absence of data on longer-term health outcomes.</p><p>Conclusions</p><p>Patients who were eligible for immediate ART had dramatically higher retention in HIV care than patients who just missed the CD4-count eligibility cutoff. The clinical and population health benefits of offering immediate ART regardless of CD4 count may be larger than suggested by clinical trials.</p></div

Vpu expression leads to a loss of wild-type but not a quadruple mutant tetherin protein steady state levels.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the absence of HIV-1 Vpu (black bar), or presence (white bar). Mutation of positively selected residues I26V, V30G, I36L, T45I (ΔTHN) in the trans-membrane region of human tetherin results in reduced sensitivity to Vpu whilst maintaining antiviral activity. The effect of co-transfection of HIV-1 plasmids and untagged human tetherin is shown for comparison (Lane C). The titre of the unrestricted HIV-1 was 10⁷ infectious units/ml. Errors are standard error of the mean of 2 experiments. (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in extracts from transfected 293T cells. Cell extract lysates (D) or pellets (F) were blotted for the Xpress tag to detect tetherin. Sizes of molecular weight markers are shown in kilodaltons. Blots in D (E) or F (G) were stripped and re-probed for β actin as a loading control. Data are representative of 3 independent experiments and similar results were seen with an N terminal HA tag.</p